Both clinical applications as well as applications in the food industry require highly efficient methods for screening samples on the presence or the absence of pathogenic micro-organisms. Whereas screening methods for clinical applications require high-throughput, fast and correct identification of the micro-organisms present in the sample for diagnostical purposes, applications in the food industry require highly reliable high-throughput methods for the microbiological control and microbiological monitoring to validate the safety and quality of food products. For both instances, methods are required that allow the fast detection, identification and characterization of micro-organisms in various samples. Other important requirements for such screening methods are the ease of use and the elimination of errors (false positives and/or false negatives).
Various screening methods known in the art such as real-time PCR enable fast screening and provide a method for testing on presence or absence of pathogenic bacteria. However, e.g., after detecting the presence of micro-organisms, further identification requires multiparameter testing, which generally cannot be provided by these screening methods. For instance, real-time PCR allows only limited multiplexing of biomarkers. In addition, detection of small changes in DNA sequences such as SNPs is often difficult.
Typical identification and characterization test methods are slow, laborious and require a high level of expertise to execute and to interpret the results. Subtyping bacteria, or determining of antibiotic resistance can only be done by classical expensive and laborious methods that require a large amount of expertise. Furthermore, the typical molecular typing methods (Ribotyping, MLST, AFLP, MLVA, MicroSeq, rep PCR etc.) require the tests to be done on pure strains. Purification of samples is often also a very tedious process, taking a long time. Many of these tests are therefore performed in specialized laboratories. The main disadvantage of this approach is that the results of the further identification of positive samples are very often too late for practical use as well as being relatively expensive, both in food and also in clinical field.
A method for efficiently screening samples on the presence or the absence of pathogenic micro-organisms and the subsequent identification of the micro-organisms has been described in EP1633887. However, a problem of the method described in EP1633887 is that all of the samples are subjected to the entire process. Even if no micro-organisms are detected, the step of screening the samples on a microarray is performed for all samples. Since generally more than 95% of food samples and water control samples are negative for the presence of micro-organisms, a lot of these samples are tested, and subjected to identification and characterization of micro-organisms which are not present in the sample. The described screening method therefore provides avoidable process steps.
In view of time and cost considerations, the present invention provides an optimized method for the detection, identification and characterization of micro-organisms in samples. The method detects in first instance whether or not micro-organisms are present in the sample and in a next step identification and characterization is carried out on the positive samples only.